The Promise of RNAi in Honey Bee Health and Research
Malcolm T. Sanford
Bee Culture (November ) Vol. 136: 15-17
This author takes some pride in bringing to the readers of this magazine various “scoops” of scientific information they might not have heard before. I have done this recently in describing both epigenetics1 and Nosema ceranae.2 Now get ready to be indoctrinated in another technology that is coming on strong called RNAi.
Gene silencing” is one of the hottest topics in science these days. It is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification and is related to epigentics, the regulation of genes through environmental factors.3 A gene that would be expressed (turned on) under normal circumstances is in essence switched off in gene silencing.
Recall that the instructions for all life processes in plants and animals are found in the double-helix molecule, deoxyribonucleic acid (DNA). The DNA is a template from which ribonucleic acid (RNA), often called messenger RNA, is formed. An enzyme, RNA polymerase delivers (transcribes) the DNA’s information onto RNA, stimulating genes to turn on or “express” themselves, usually by producing certain proteins, the chemical building blocks of life. If the message is not delivered, the gene is not turned on. In summary, the life process that is supposed to occur never begins, because the proper message is not received. The DNA genetic information has been “silenced.”
RNA interference (RNAi) is a major cause of gene silencing in organisms. The term was coined by two scientists, who were awarded Nobel Prize in Physiology or Medicine in 2006. The work of Craig C. Mello and Andrew Fire capped a long history in genetic analysis in plants and other organisms investigating genetic expression and suppression. Their study of RNAi in nematodes (round worms) revealed a mechanism responsible for gene-silencing. The exact way RNAi works is complex, but begins when double-stranded RNA is created, then introduced into a cell, which recognizes it as somewhat of a foreign substance, and this activates RNAi.4
The ability to silence genes brings to the table huge possibilities in all realms of genetic investigation. RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants. In a previous column,5 I discussed the role of viruses in honey bees health. One of the statements I wrote at the time was “Besides being inconspicuous to beekeepers, other reasons exist for the relatively little work on honey bee viruses by scientists over the years. They are not easy to detect, and even if there was any evidence viruses were doing harm to honey bee colonies, there were few if any treatment options.” The former topic of detection is becoming revolutionized via new technologies like those I mentioned in that column, including the RT-PCR assay and the Integrated Virus Detection System (IVDS).
We can now look at the latter topic, what to do about viral infections after they are detected, with a fresh eye thanks to efforts of scientists looking at possibilities using RNAi. Viruses produce their own messenger RNA, which hijacks the host’s cell mechanics to replicate the virus instead of the original organism or host. If this can be interfered with via RNAi, the viral RNA no longer can do its job. Because the genome of viruses is shorter and less complicated than other organisms, it becomes relatively easier to get their DNA sequenced. A pioneer firm in this area is a brand new startup company that beekeepers no doubt will be hearing more about called Beeologics. It has the motto, “Working together to address the bee crisis.”
According to its web site,6 “Beeologics is an international firm focused on restoring bee health and protecting the future of insect pollination. With offices and laboratories in the United States and Israel, Beeologics brings together some of the world’s most recognized virologists and microbiologists and is the only company to make a scientific breakthrough on the road to preventing bees from succumbing to viruses.”
“Incorporated in 2007, Beeologics founded upon many years of research conducted by some of the world’s leading scientists. It is due to this unique integration of diversified talent that Beeologics has quickly commanded worldwide attention and is recognized by the USDA, Department of Agriculture, and leading entomologists.
“While its primary focus remains on overcoming the CCD crisis, Beeologics’ mission is to become the guardian of bee health worldwide. Through innovation, continuous research, and focus on applicable solutions, Beeologics is developing a line of products specifically addressing the bee’s well being.”
The first product Beeologics is attempting to bring to the market is Remebee®. The advantages of this include the following:
Potent protection from Israel Acute Paralysis Virus (IAPV)
Potentially applicable to all bee viruses
Inherent robustness precludes possibility of virus breaking resistance
Extreme specificity and no toxicity
No residues in honeybees or honey
It would seem that the folks at Beeologics are hanging Remebee’s® hat on its protection from Israeli acute paralysis virus (IAPV), which was given wide publicity at the World Apicultural Congress in Melbourne, Australia (Apimondia 2007) as being strongly correlated with CCD. This provoked a controversy that I have discussed in a previous column on bee viruses. Remebee® which is stated as the first line of defense product is the stated foundation for the development of a comprehensive RNAi based anti viral agent for a series of known bee viruses.
Beeologics has begun the regulatory approval process for Remebee® through the Federal IR4 agency to register it as a biopesticide, by the Environmental Protection Agency (EPA).7 A recent ruling determined that it needs to be registered as bee medicine with the Department of Veterinary Medicine in the Food and Drug Administration (FDA)8. This process is accompanied by the National Research Support Project No. 7 (NRSP-7)9 which was established by the USDA and FDA to address the shortage of minor species animal drugs by funding and overseeing the efficacy, animal and human safety, and the environmental assessment required for drug approval. Currently there are only five active agents registered as therapeutic drugs for use on honeybees at the FDA, none of which are based on RNAi technology.
Although the timeline for registering a new medicine for a minor species is generally considered shorter than major food animals or human therapeutic drugs, it still must go through a rigorous process. Furthermore, the RNAi technology is so new that many questions still remain about registering materials developed using double-stranded RNA including the registering agency. Nevertheless, the firm is going forward with what it is calling a “clinical trial” and is signing up beekeepers as cooperators, the goal being to include over 100,000 hives. This would no doubt be one of the largest bee research projects of its kind ever to take place in the United States. Depending on how this shakes out, the folks at Beeologics may be asking beekeepers and scientists to help in this regard by enlisting their aid in informing the regulators that a genuine honey bee emergency exists in the form of colony collapse disorder (CCD), which is in fact correlated with Israeli acute paralysis virus (IAPV).
In the meantime, Beeologics is going ahead with another product called RemebeePlus®, a novel and unique feed formulation. This feeding supplement would be based on natural ingredients and does not appear to require much regulatory scrutiny.
The RNAi technology is new and expensive to research. So far Beeologics has held trials for Remebee® in both Florida and Pennsylvania and published papers from these studies are expected soon. At the same time, the firm has publicized its work in Europe and developed close relationships with researchers there as well. The final cost of a product of this nature is of concern. There is little doubt that the firm is investing a good deal of venture capital in this enterprise and so will have to balance its potential revenue against these expenses. No firm price has yet been established for any of its products.
Beyond the work by Beeologics, the technology will continue to be one examined by others in the field. Dr. Eric Mussen at the University of California, Davis has written an analysis of the potential of the technique in his most recent newsletter, From the UC Apiaries.10 He concludes: “If we can find a way to stimulate the anti-viral response in bees, we should ask the molecular biologists to find out what genes are involved so that we can survey our stocks and see which ones respond best to stimulation. We should favor those stocks in future selection programs.”
The new hire also at the University of California, Davis (Haagen-Dazs Post Doctoral Fellow Michelle Flenniken) also expects to focus on RNAi. According to a press release, “Skilled in multidisciplinary research--molecular biology, microbiology, chemistry and cell biology, she will study the biology of honeybee viruses, specifically the role of RNA interference in the honey bee antiviral immune responses. She has been quoted that this technology can be used as an antiviral strategy in honey bees, and will investigate the role of the RNAi machinery in virus infection and attempt to limit virus production in the bees by priming their RNAi machinery with viral specific double-stranded RNA.”11
It is a given that in order to silence a gene, researchers must have the organism’s genome to work with. The Remebee® RNAi solution requires intimate knowledge of both the specific viral and honey bee genome. This may not be at first obvious for it is possible to get appropriate genetic information from the virus and not knowing the hosts.
The a question about the applicability in the case of Remebee® was answered in an e-mail from the firm, “In order to design double stranded RNA sequences that are specific only to the virus, we BLASTED (sequence comparison using robust software tools) the bee genome to make sure that none of the proposed Remebee® sequences bears more than 20 b.p. sequences homologous (identical) to the honeybee genome. This is not only important to avoid inadvertently silencing a possibly important bee gene, but also because it helps regulating authorities declare the treatment safe. By the way, we did the same vs. the human genome to ensure the FDA that if accidentally swallowed by the beekeeper it cannot inadvertently silence human sequences (Although this is extremely unlikely even if there were homologous sequences).”
It is extremely fortunate that the Honey Bee Genome Project (HBGP) that I discussed in earlier columns12 has been completed or applicable safety concerns could not have been addressed. Thanks must go Dr. Gene Robinson and colleagues who developed the white paper that was the basis for the DNA sequencing. In those articles, I listed the reasons for and potential expected results for supporting the HBGP, including the following:
“Mental health. Some forms of mental illness, such as autism, involve problems with social integration. Bees show a high degree of social integration, and their activities are highly dependent upon their ability to read social cues; identification of several well-defined sets of social cues make for unusually tractable experimental social systems.
“Biosensors. A HBGP also may enhance use of honey bees as environmental sentinels.
“Instincts. The societies of honey bees and other social insects occupy Wilson’s second “pinnacle of social evolution,” with complexity that rivals our own. Among the provocative similarities are: extensive communication systems (including the only non-primate symbolic language); highly organized defense and warfare; complex architecture (including the insect equivalent of skyscrapers – 4 meter high termite nests in Africa); and expressions of personal sacrifice unheard of in most of the rest of the animal kingdom.
“Cognition. Bees collect food from flowers, a highly ephemeral food source, and have evolved sophisticated cognitive abilities to maximize foraging success. They are excellent at associative learning, based on the need to associate a color, shape, scent, or location with a food reward. Honey bees also can learn abstract concepts such as "similar" and "dissimilar," and are able to negotiate complex mazes by using visual stimuli as direct or abstract "signposts" or by recognizing path irregularities.
“Gerontology. Queens and their workers have identical genotypes but queens live two orders of magnitude longer. Identification of all differentially expressed genes responsible for these striking differences in lifespan, facilitated by a HBGP, undoubtedly has important implications for human longevity and aging.”
Nowhere in the above panoply is the mention of RNAi. However, like so many new technologies, this specific application may not have been on the radar screen of those proposing the HBGP project. Thus, like the development of Teflon® and other developments due to the U.S. space program, those looking at RNAi and its use in honey bee research have a new tool thanks to the honey bee genome project to help them sort out the various challenges affecting beekeepers. Most researchers I’ve talked to say this technology has lots of promise in helping beekeepers address some of the most challenging problems facing their industry. Let’s hope they are right.
References: All URLs accessed September 19, 2008.
1. Sanford, M.T. 2008, “More Than Just
the Honey Bee Genome: Does Epigenetics Play a Role?” Bee
Culture, Vol. 136: 17-19 (May) see
2 Sanford, M.T. 2007. A New Nosema, Bee Culture Vol. 135 (2): 18-21 (February). See: http://www.squidoo.com/Bee_Culture
5. Sanford, M.T. 2008, “Viruses and Honey Bees,” Bee Culture, Vol. 136, No. 8, pp. 19-20 (August) see http://www.squidoo.com/Bee_Culture
10. Mussen, E. 2008. From the UC Apiaries, http://entomology.ucdavis.edu/faculty/mussen/JulAug2008.pdf
12. Sanford, M.T. 2006. “Update on the Honey Bee Genome Project,”
Bee Culture, Vol. 134 (12): 21-24 (December), and Sanford, M.T. 2003. “Cracking the Honey Bee’s Genetic Code,”Bee Culture (April 2003), Vol. 131 (4): 19-21, see http://www.squidoo.com/Bee_Culture